The objective of this research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions In the delivery of newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. A key step in this pathway is the selective phosphorylation of mannose residues on the high mannose glycans of the acid hydrolases by UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (Ptase). This transferase is an alpha2betagamma2 hexameric protein encoded by two genes. We established that the alpha/beta subunits recognize the common protein determinant of the acid hydrolases as well as mediating the catalytic function of the enzyme. The y subunit enhances the activity of the alpha/beta subunits toward a subset of the acid hydrolases. Specific Aim 1 is directed toward identifying the domain of the alpha/beta subunits that binds the protein determinant of the acid hydrolases. Aim 2 focuses on the role of the Man-6-P receptor homology (MRH)-domain of the gamma subunits in enhancing the activity of the a/p toward selected hydrolases. Aim 3 seeks to understand the physiologic basis for the development of neurodegeneration in alpha/beta null mice. Aim 4 is to determine how Ptase is localized to the cis-Golgi subcompartment. Aim 5 is to define the essential role of the GGA2 (for Golgi-localized, y-ear containing, ARF-binding) coat protein in maintaining neonatal viability. This function cannot be replaced by GGA1 and GGA3, establishing that the GGAs are not fully redundant. These studies will utilize mice with disruption of the genes encoding the three GGAs.